- Title
- An examination of how environmental disturbances alter microglia morphology and function within the CNS
- Creator
- Kongsui, Ratchaniporn
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2017
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Almost daily the role number of roles that microglia have been identified as playing a role in expands. Once considered to only act as the macrophages of the central nervous system, microglia are now recognised to be ‘master-orchestraters’ of events as diverse as neurogenesis, synaptic pruning, synaptogenesis, vascular remodeling, extracellular matrix remodeling, vascular surveillance and many others. Perhaps one of the most interesting and enigmatic features of microglia is their capacity to engage in rapid and profound structural reorganization. This ability has thus fascinated many researchers, a phenomenon that is only continued to grow in response to recent real time imaging of the cells in action. Most of is known about microglial structure has come from static two-dimensional images. Detailed morphological characterization of these cells in two dimensions, while significantly less glamorous, than real time multiphoton imaging has nevertheless proven to be an extremely reliable vehicle for revealing and characterizing the fundamental properties of state changes of these cells. Under normal conditions microglia are recognised to exist in the brain with relatively small somas and a number of thin tapering branches radiating out from the cell body. Often, although not always, these branches split and often split again in what is frequently referred to as their secondary and tertiary branching structures. Following exposure to severe environmental disturbances, microglia are known to engage in profound structural reorganization. The specific form that this reorganization takes is somewhat interventions specific but in many cases it will involve marked somal enlargement and significant process simplification and shortening. These changes are ultimately considered to be relevant and informative because they are known to be linked with changes in the specific functions engaged in by microglia. Chapter 2: At the outset of my studies there had been no quantitative studies to what represented ‘normal’ microglial morphology within uninjured prefrontal cortex (PFC). The PFC is well documented to play a critical role in mood disturbance and our research group has found evidence that chronic stress induces significant changes in microglial activity in this region. Accordingly, the major aim of Chapter Two was to systematically examine the morphology of microglia (identified by ionized calcium-binding adaptor molecule-1 (Iba-1) labelling). Here I undertook many hundreds of manual reconstructions across the five cortical layers of the rat PFC. The reconstruction analysis was used to determine the morphometric parameters including the convex hull area, cell body perimeter, total length, total volume, number of processes, number of branch points, and the complexity of cell. Our results revealed that microglial morphology was consistent across all layers by demonstrating as ramified microglia, while there was a variation in cell size. I additionally determined the density of microglia and found no difference in anterior-posterior direction of the PFC. These findings confirmed that microglia represent a relatively homogenous within the healthy PFC. Chapter 3: Several previous studies have linked expression of the purine ionotropic receptor P2X7, which is primarily found on microglia, with major depression. As chronic stress is recognised to be a significant risk factor in the emergence of major depression. I decided to identify if chronic restraint stress was involved in changes in the expression of P2X7 within the hippocampus. In this study, I utilized an analysis technique recently developed by our group known as cumulative threshold spectra (CTS) to identify if stress altered P2X7 at any of three different time points: acute stress (1 session), chronic stress (21 sessions) and a chronic stress with 7 days recovery time. The findings of this work suggested that exposure to acute or chronic stress significantly reduced the expression of P2X7 receptor within the hippocampus and that levels remained suppressed in recovery period. In addition, the comparative analysis on normalised data showed that the level of the P2X7 reduction was significantly greater in the chronic stress relative to the acute stress group. Furthermore, I found that P2X7 expression rebounded to baseline in 7 days recovery time. Collectively, these results provide the first evidence that chronic restraint stress produces markedly a reduction of the P2X7 receptor within the hippocampus. Chapter 4: In Chapter Four, I investigated how chronic stress influences the roles of glial cells (microglia and astrocytes) as it related to disturbances of glutamatergic signaling. Using immunohistochemical analysis, the results showed that there was a reduction in glial fibrillary acidic protein (GFAP), a marker of astrocytes, in the hippocampus after exposure to chronic stress, while the expression of Iba-1 was increased. In addition, I examined how these changes were associated with glutamatergic signalling proteins following chronic stress. Specifically I assessed changes in the expression of the astrocyte specific glutamate transporters EAAT1 and EAAT2. The proteins were observed to have opposition changes after stress with a increase in EAAT1 and a decrease in EAAT2 expression. Further, I measured the expression of related proteins such as vesicular glutamate transporter (VGLUT1), which was increased and AMPA and NMDA subunits (GluA1, GluA2, GluN1, GluN2A, GluN2B). The AMPA and NMDA subunits showed a complex pattern of changes, however, when taken together with the changes in VLGUT1 and EAATs 1 and 2 are strongly suggestive of dramatic shift in glutamatergic activity. Together, these findings suggest that chronic stress-induced structural remodelling of astrocytes and microglia may be driven by alterations in glutamatergic neurotransmission. Chapter 5: Besides the roles of microglia in response to chronic stress-induced mood disorder, previous studies have demonstrated that microglia appear to be the most important cell involved in inflammatory related mood disturbances. The administration of lipopolysaccharide (LPS) is well-validated model of immune challenge producing microglial activation and sickness behaviours in rodents and humans. Recently, these behaviours have become the representative indicator of how peripheral disturbances in immune signalling can disturb quite complex behaviours. Therefore, I examined the alterations of microglial structure within the PFC following the administration of LPS (100 µg/kg/i.p.) in adult male rats. Immunohistochemistry was conducted to analyse the changes in Iba-1, a specific microglial marker, in the infralimbic and prelimbic of the PFC using a cumulative threshold spectra (CTS) analysis and digital reconstructions. Our results indicated that the somal area of was significantly enlarged, while cell processes became slightly shortened after LPS injection. This data is the first to demonstrate significant microglial alterations within the PFC following LPS administration. Collectively, the experiments clearly outline the complex nature of the structural changes that microglia can undergo in response to routine, yet severe, environmental challenges.
- Subject
- microglia; chronic stress; neuroinflammation; Iba1
- Identifier
- http://hdl.handle.net/1959.13/1336107
- Identifier
- uon:27548
- Rights
- Copyright 2017 Ratchaniporn Kongsui
- Language
- eng
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| Thumbnail | File | Description | Size | Format | |||
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| View Details Download | ATTACHMENT01 | Thesis | 13 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 893 KB | Adobe Acrobat PDF | View Details Download |